Pesticidal Nucleic Acids and Proteins and Uses Thereof

ABSTRACT

The invention provides compositions comprising polynucleotide molecules encoding certain pesticidal polypeptides which exhibit plant parasitic nematode and/or insect control properties, and are particularly directed to controlling plant parasitic pest species of nematodes and insects known to infest crop plant species. Methods for controlling pests are disclosed in which the toxic proteins are provided in the diet of the targeted plant pests. The invention also provides compositions such as nucleic acids, proteins, and plant and bacterial cells, plants, and seeds containing the nucleic acid and protein compositions, as well as methods and kits for identifying, detecting, and isolating the compositions of the present invention. The invention further provides a method of producing crops from recombinant seeds which contain the polynucleotide molecules encoding the pesticidal polypeptides of the present invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 61/441,697 and U.S. Provisional Application Ser. No. 61/441,709,both filed Feb. 11, 2011, which are incorporated herein by reference.

INCORPORATION OF SEQUENCE LISTING

The Sequence Listing accompanying this application is contained withinthe computer readable file “38-21(57560) SEQUENCE LISTING_ST25.txt”submitted electronically and contemporaneously with the filing of thisapplication through the USPTO EFS-Web. The file is 105 kilobytes(measured in MS-Windows), was created on 19 Jan. 2012, and isincorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to novel polynucleotide and protein compositionsthat, when expressed and or produced in plants, impart resistance toplant pathogenic nematodes and insect infestation. The polynucleotidesand proteins can be expressed in plant and bacterial cells, and theplant cells can be regenerated into transgenic (recombinant) plants,plant tissues, plant parts, and seeds. Compositions derived from suchplants, plant materials, and seed that contain detectable amounts ofsuch polynucleotides and proteins are included within the scope of theinvention. The invention also relates to compositions and methods forcontrolling plant pathogenic nematodes and insect pests of crop plants.

BACKGROUND OF THE INVENTION

The increasing human population will require higher yields of food,feed, and fiber from crop plants on decreasing amounts of arable land.Several types of insects and nematodes are known to reduce yield ofcrops produced from plants. Plant pests damage plant parts, includingroots, developing flower buds, flowers, leaves, stems, and seeds, whichleads to lower yields.

Traditional approaches for controlling plant pests have used chemicalcontrol agents and construction of inter-specific hybrids between cropsand their wild-type relatives as sources of resistant germplasm.Chemical pest control agents, although effective, have severaldisadvantages. Many chemical control agents are expensive tomanufacture, and are characterized as pollutants because they persist inthe environment as a result of their resistance to microbialdegradation. Chemical control agents require on-farm formulation, whichincreases the safety risk to the farmer due to the exposure to chemicalagent formulations. The chemical agent formulations have to be appliedat least once and often, more than once per growing season, increasingthe carbon footprint related to these compositions. Methods andcompositions employing plant biotechnology pest control agents are alsoeffective means for controlling plant pests, for instance through plantexpression of one or more pest control agents that are generallyselectively toxic to a particular target pest when ingested by the pest.Unlike chemical agents, biotech approaches have been demonstrated to beenvironmentally friendly, have no known safety risks when used byfarmers, and are economical in terms of carbon footprint impact and easeof use for deployment by the farmer. However, there are only a fewexamples of such biotech compositions and methods for controlling suchpests, and even fewer if any examples of any biotechnology approachesthat have demonstrated efficacy in controlling plant pathogenicnematodes. Thus, there is a need for new compositions and methods forprotecting plants from such pest infestation, generally for the purposeof maintaining and enhancing yields of crops produced from such plants,and for sustaining and providing food, feed and fiber for the increasinghuman population.

SUMMARY OF THE INVENTION

Polynucleotide molecules are provided encoding exemplary pesticidalpolypeptides as set forth in SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:30, SEQ IDNO:34, SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:50, SEQ IDNO:54, SEQ ID NO:58, and SEQ ID NO:60. Polypeptides having an amino acidsequence exhibiting from at least about 45% to about 99.9% identity tothe pesticidal protein (polypeptide) sequences as set forth in any ofthe foregoing protein sequences (any percentage in between 45 and 99.9)and exhibiting substantially equivalent (biologically functionalequivalent) pesticidal activity as any one of these sequences arespecifically contemplated. Fragments of these polypeptide sequences thatexhibit the requisite pesticidal activity are intended to be within thescope of the present invention. Such polynucleotides may be extractedand/or obtained directly from a host cell or made artificially throughvarious means of synthesis, and in either case, are considered to berecombinant polynucleotides.

Polynucleotides containing one or more nucleotide sequence segmentsencoding the pesticidal proteins of the present invention are provided,which may be operably linked to a heterologous promoter that initiatesexpression of the sequence region in a designated host cell, resultingin the production or manufacture of the pesticidal protein in the hostcell. The promoter may include a plant-expressible promoter, a promoterthat functions in one or more species of bacteria, and a yeastfunctional promoter, or combinations thereof. The plant-expressiblepromoter may include any number of promoters known in the art, includingbut not limited to corn sucrose synthetase 1 promoter, corn alcoholdehydrogenase 1 promoter, corn light harvesting complex promoter, cornheat shock protein promoter, pea small subunit RuBP carboxylasepromoter, Ti plasmid mannopine synthase promoter, Ti plasmid nopalinesynthase promoter, petunia chalcone isomerase promoter, bean glycinerich protein 1 promoter, Potato patatin promoter, lectin promoter, CaMV35S promoter, FMV promoter, ubiquitin promoters promoter, and the S E9small subunit RuBP carboxylase promoter.

Isolated polynucleotide segments are provided for use as probes and/orprimers, which may be from about 20 to about 1000 contiguous nucleotidesin length or any length in between twenty and one thousand contiguousnucleotides, and exhibit at least about 90% identity to the samecontiguous length of nucleotides as set forth in any of SEQ ID NO:1, SEQID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ IDNO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ IDNO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ IDNO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ IDNO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, and SEQID NO:63, or the complement of any of the foregoing polynucleotidesequences.

In another aspect of the invention, polynucleotides encoding any of thepesticidal polypeptides set forth above are provided in recombinantexpression cassettes. The expression cassettes can be provided invectors for use in replicating, maintaining and transferring the nucleicacid component encoding the pesticidal proteins of the presentinvention. The vectors of the present invention contain at least asequence region that encodes the polypeptide as set forth above. Thevector includes a plasmid, baculovirus, artificial chromosome, virion,cosmid, phagemid, phage, or viral vector.

Host cells may be any appropriate transgenic host cell including but notlimited to microbial cells (microorganisms) such as an Agrobacterium, aBacillus, an Escherichia, a Salmonella, a Pseudomonas, a Rhizobiumbacterial cell, a yeast cell such as a pichia yeast or saccharomycesspecies yeast cell, or a plant cell. Vectors as described above can beprovided in a transgenic microbial host cell. The transgenic microbialhost cell includes a prokaryotic or eukaryotic host cell. The transgenicprokaryotic host cell is a bacterial cell and the transgenic eukaryotichost cell is a plant or a fungal/yeast cell. The transgenic bacterialcell includes a recombinant bacterium including a Bacillusthuringiensis, Bacillus subtilis, Bacillus megaterium, Bacillus cereus,Bacillus laterosperous, Escherichia, Salmonella, Agrobacterium,Rhizobium, or Pseudomonas cell. The transgenic plant host cell includesa monocotyledonous or dicotyledonous plant cell and may include anyplant cell from the Group of Plants or Plant Group set forth below. Tothe extent that a microbial cell is a plant cell, the cell can beobtained from any plant, plant tissue, plant part or seed from a plantselected from the group consisting of any of the following, includingbut not limited to barley, bean, broccoli, cabbage, canola (rapeseed),carrot, cassava, castor, cauliflower, celery, chickpea, Chinese cabbage,coffee, corn (including sweet corn), clover, cotton, a cucurbit,cucumber, deciduous trees (including but not limited to banana, citrus,eucalyptus, nut trees (including but not limited to hickory, pecan, andwalnut trees), oak trees (including but not limited to live oak, pinoak, and post oak trees), olive, palm (including coconut palm), poplar,sweet gum, and rootstocks of all of the preceding trees), eggplant,evergreen trees (including but not limited to Douglas fir), flax,garlic, grape, grasses (including but not limited to alfalfa, pasturegrass, switchgrass, and turf grass), hops, leek, lettuce, millets,melons (including but not limited to cantaloupe, honeydew melon, andwatermelon), oat, onion, pea, peanut, pepper, pigeonpea, pine (includingLoblolly pine, Radiata pine, and Southern pine), potato, pumpkin,radish, rice, rye, safflower, shrub, sorghum, soybean, spinach, squash,strawberry, sugar beet, sugarcane, sunflower, sweet corn, sweet potato,tea, tobacco, tomato, triticale, or wheat. The aforementioned arereferenced herein as the “Group of Plants” or the “Plant Group”.

Recombinant plants, plant tissue, plant parts, or seed contain thepolynucleotides of the present invention and express the proteins of thepresent invention from such polynucleotides. The plant part is a leaf, astem a flower, a sepal, a fruit, a root, or a seed. Products producedfrom a recombinant plant of the present invention are also contemplated,and can include at least any of the following: oil, meal, lint and seedof the recombinant plant. The polynucleotides and proteins of thepresent invention are present in a detectable amount in the plants andplant products, and are useful at least as markers for tracking thepresence of seeds and plant tissues containing the polynucleotide andproteins through trade and commerce, in fields of crops, and in variousembodiments referenced herein.

There is provided a method of detecting and/or isolating in or from abiological sample, a polynucleotide molecule encoding a pesticidalpolypeptide of the present invention in which the steps of the methodinclude (i) selecting a pair of oligonucleotide primers that produce anamplicon encoding all or a representative amount of the pesticidalpolypeptide of the present invention when used together in anamplification reaction with the biological sample containing thepolynucleotide; (ii) producing the amplicon from the polynucleotide;(iii) detecting and/or isolating the amplicon; and (iv) generatingnucleotide sequence information corresponding to the amplicon toidentify and confirm the presence (or absence) of a segment of apolynucleotide molecule encoding all or a representative amount of thepesticidal polypeptide. Alternatively, the detecting and/or isolatingstep can be conducted by providing a polynucleotide probe derived from asufficient length of DNA or RNA encoding the pesticidal polypeptide thathybridizes under specific or under stringent hybridization conditions tosuch a polynucleotide encoding a pesticidal polypeptide of the presentinvention.

Methods of controlling or killing a target lepidopteran pest,coleopteran pest, or plant pathogenic nematode pest population areprovided and include contacting the pest population with apesticidally-effective amount of the polypeptide as set forth above. The“lepidopteran pest population” includes Spodoptera frugiperda,Spodoptera exigua), Mamestra configurata, Agrotis ipsilon, Trichoplusiani, Pseudoplusia includens, Anticarsia gemmatalis, Hypena scabra,Heliothis virescens, Agrotis subterranea, Pseudaletia unipuncta, Agrotisorthogonia, Ostrinia nubilalis, Amyelois transitella Crambuscaliginosellus, Herpetogramma licarsisalis, Homoeosoma electellum,Elasmopalpus lignosellu, Cydia pomonella, Endopiza viteana, Grapholitamolesta, Suleima helianthana, Plutella xylostella, Pectinophoragossypiella, Lymantria dispar, Blatta orientalis, Blatella asahinai,Blattella germanica, Supella longipalpa, Periplaneta americana,Periplaneta brunnea, Leucophaea maderae, Alabama argillacea, Archipsargyrospila, A. rosana, Chilo suppressalis, Cnaphalocrocis medinalis,Crambus caliginosellus, C. teterrellus, Diatraea grandiosella, D.saccharalis, Earias insulana, E. vittella, Helicoverpa armigera, H. zea,Heliothis virescens, Herpetogramma licarsisalis, Lobesia botrana,Pectinophora gossypiella, Phyllocnistis citrella, Pieris brassicae, P.rapae, Plutella xylostella, Spodoptera exigua, S. litura, S. frugiperda,Tuta absoluta. The “coleopteran pest population” includes Anthonomusgrandis, Lissorhoptrus oryzophilu, Sitophilus granaries, Sitophilusoryzae, Hypera punctata, Sphenophorus maidis, Leptinotarsa decemlineata,Diabrotica virgifera virgifera, Diabrotica barberi, Diabroticaundecimpunctata howardi, Chaetocnema pulicaria, Phyllotreta cruciferae,Colaspis brunnea, Oulema melanopus, Zygogramma exclamationis, Epilachnavarivestis, Popillia japonica, Cyclocephala boreali, Cyclocephalaimmaculata, Rhizotrogus majalis, Phyllophaga crinita, Ligyrus gibbosus,Melanotus spp., Conoderus spp., Limonius spp., Agriotes spp., Cteniceraspp., Aeolus spp., Eleodes spp. The “plant pathogenic nematodepopulation” includes Heterodera glycines (soybean cyst nematode),Heterodera schachtii (beet cyst nematode), Heterodera avenae, Globoderarostochiensis, Globodera pailida, Pratylenchus zeae (a root knotnematode), Meloidogyne javanica, Pratylenchus brachyurus (a root knotnematode), Meloidogyne hapla, Meloidogyne incognita.

An alternative method for controlling such plant pest infection includesproviding a pest inhibitory amount of a pesticidal polypeptide of thepresent invention to a pest susceptible to the polypeptide, therebycontrolling the pest. The pest is an insect or a nematode. The insectmay be any insect within the taxonomical orders including Coleoptera,Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera,Orthoptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera,or Trichoptera (hereinafter, the “Insect Orders”). The nematode may befrom any genus of nematodes referred to as Acontylus, Anguina,Aorolaimus, Aphasmatylenchus, Aphelenchoides, Aphelenchus, Atalodera,Atylenchus, Bakemema, Belonolaimus, Brachydorus, Bursaphelenchus,Cacopaurus, Caloosia, Carphodorus, Criconema, Criconemella, Cryphodera,Ditylenchus, Dolichodorus, Eutylenchus, Globodera, Gracilacus,Helicotylenchus, Hemicriconemoides, Hemicycliophora, Heterodera,Hirschmanniella, Histotylenchus, Hoplolaimus, Hoplotylus, Longidorus,Macrotrophurus, Meloidodera, Meloidogyne, Merlinius, Morulaimus,Nacobbus, Nothanguina, Nothotylenchus, Paralongidorus, Paratrichodorus,Paratrophurus, Paratylenchus, Peltamigratus, Pratylenchoides,Pratylenchus, Psilenchus, Radopholoides, Radopholus, Rhadinaphelenchus,Rototylenchus, Rotylenchoides, Rotylenchus, Sarisodera, Scutellonema,Sphaeronema, Subanguina, Telotylenchoides, Telotylenchus,Trichotylenchus, Trophonema, Trophotylenculus, Trophurus,Tylenchorhynchus, Tylenchulus, Tylenchus, Tylodorus, Xiphinema, orZygotylenchus (hereinafter, the “Nematode Species”). In relatedembodiments, the nematode species includes cyst and related nematodessuch as Heterodera glycines (soybean cyst nematode), Heteroderaschachtii (beet cyst nematode), Heterodera avenae (cereal cystnematode), and Globodera rostochiensis and Globodera pailida (potatocyst nematodes), Pratylenchus zeae, Meloidogyne javanica, Pratylenchusbrachyurus, Meloidogyne hapla, or Meloidogyne incognita (hereinafter,the “Cyst Nematode” group). The pest inhibitory amount of the pesticidalpolypeptide is provided in the diet of the pest, and the diet of thepest can be a part of a recombinant plant, seed of such plant, orproduct of the plant. The pest inhibitory amount of the polypeptide mayalso be provided in a topical formulation to a plant. Such formulationcould include a preparation containing bacterial cells, bacterialspores, and parasporal crystals which contain or are producing one ormore of the polypeptides/toxic agents of the present invention in asufficient amount to inhibit the pest infestation of the plant to whichthe formulation is applied. A formulation for controlling nematode orinsect species within the scope of the present invention may consist ofrecombinant bacterial cells and/or sporeswhich may be producing thetoxic proteins of the present invention, or parasporal crystals thatcontain pesticidal amounts of the polypeptide. The bacterial cells,spores, or parasporal crystals are typically from Bacillus species.Antibodies are contemplated that specifically bind to a polypeptidehaving the amino acid sequence as set forth in any of SEQ ID NO:2, SEQID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ IDNO:26, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:42, SEQ IDNO:46, SEQ ID NO:50, SEQ ID NO:54, SEQ ID NO:58, and/or SEQ ID NO:60, ora peptide, or an epitope derived therefrom. Particularly, purifiedantibodies that specifically bind to one or more of the polypeptides ofthe present invention, or to a peptide or epitope derived from theproteins of the present invention are contemplated.

Such antibodies are useful at least in methods of detecting pesticidalpolypeptides such as those set forth in SEQ ID NO:2, SEQ ID NO:6, SEQ IDNO:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ IDNO:30, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:46, SEQ IDNO:50, SEQ ID NO:54, SEQ ID NO:58, and/or SEQ ID NO:60 in a biologicalsample. A method of detecting such proteins could include the steps ofcontacting the biological sample with an antibody that specificallybinds to one or more of the proteins of the present invention, anddetecting the binding of the antibody to the pesticidal polypeptide.Alternatively, proteins of the present invention, or proteins that aresubstantially related to the proteins of the present invention can bedetected in or isolated from a biological sample either by directlyidentifying the protein in the sample using for example, antibodies asindicated above, or by screening for the presence of a polynucleotideencoding the pesticidal protein. Detecting the polynucleotide encodingsuch protein could include the steps of: i) selecting a pair of primersthat produce an amplicon encoding the pesticidal protein when usedtogether in an amplification reaction with the polynucleotide; ii)producing the amplicon by using the polynucleotide as a template in theamplification reaction; iii) detecting/isolating the the amplicon; iv)generating DNA sequence information corresponding to the amplicon toconfirm that the amplicon encodes the pesticidal protein; and v) testingthe pesticidal protein to confirm pesticidal activity. Alternatively, amethod for detecting the protein of the present invention, or a relatedpesticidal protein such as a δ-endotoxin polypeptide, in a biologicalsample could include the steps of: i) obtaining a biological samplesuspected of containing a δ-endotoxin polypeptide; ii) contacting thesample with an antibody that specifically binds to the polypeptide underconditions effective to allow the formation of immune complexes; andiii) detecting the immune complexes so formed. Another alternativemethod for detecting a target pesticidal polypeptide of the presentinvention in a sample may include the steps of: i) contacting the samplewith an antibody that specifically binds the target pesticidalpolypeptide; ii) detecting the binding of the antibody to the target inthe sample; and iii) identifying the target as a pesticidal polypeptideexhibiting at least 90% amino acid sequence identity to any one of theproteins set forth in SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ IDNO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:30, SEQ IDNO:34, SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:50, SEQ IDNO:54, SEQ ID NO:58, and/or SEQ ID NO:60.

Detection methods can be conducted using reagents and instructionspackaged together in kit form and are useful for detecting the proteinsand polynucleotides of the present invention. Such kits could include afirst reagent or antibody that binds specifically to the polypeptide, orspecifically to a peptide or an epitope derived therefrom; and a secondreagent such as a control polypeptide corresponding to any of theproteins as set forth in any of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10,SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:30,SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:50,SEQ ID NO:54, SEQ ID NO:58, and/or SEQ ID NO:60, or a peptide, or anepitope derived therefrom.

In another aspect of the present invention, there is provided a methodof preparing insect resistant plants. Such plants can be prepared bycontacting a recipient plant cell with a transgene that encodes one ormore of the polypeptides of the present invention under conditionspermitting the uptake of the transgene by the cell, and selecting arecipient cell in which the transgene has been incorporated into thecell genome, and regenerating a plant from the selected recipient cell.The regenerated plant is confirmed to be a fertile transgenic plantexhibiting pest resistance, and the pest resistance includes resistanceto plant pathogenic nematode infestation and one other pest resistanceselected from resistance against to a coleopteran insect or to alepidopteran insect. The contacting step includes any one or ore of themethods known in the art, including microprojectile bombardment,electroporation or Agrobacterium-mediated plant cell transformation. Theregenerated plant is resistant to at least one of the members of theplant parasitic nematode group including Heterodera species, Globoderaspecies, Meloidogyne species, Rotylenchulus species, Hoplolaimusspecies, Belonolaimus species, Pratylenchus species, Longidorus species,Paratrichodorus species, Ditylenchus species, Xiphinema species,Dolichodorus species, Helicotylenchus species, Radopholus species,Hirschmanniella species, Tylenchorhynchus species, or Trichodorusspecies.

Transgenic seed containing one or more polynucleotide segments encodingone or more of the proteins of the present invention may be producedcomprising the steps of: transforming a plant with a transgene thatencodes the polypeptide as set forth above, the transgene operablylinked to a promoter that expresses the transgene in a plant, therebyobtaining a fertile transgenic plant comprising the transgene; andgrowing the plant under appropriate conditions to produce the transgenicseed.

Progeny of any generation of a pest resistance-enhanced fertiletransgenic plant can be produced from such transgenic plants and seedsof the foregoing plants and seed, wherein the progeny contain thepolynucleotide and encode the protein(s) of the present invention, andhas enhanced pest resistance against a coleopteran insect, lepidopteraninsect, or a plant pathogenic nematode relative to the correspondingnon-transgenic plant.

Pest resistant plants can be produced by following the method of: (a)crossing a pest resistant plant comprising a transgene that encodes thepolypeptide as set forth above with another plant; (b) obtaining atleast one progeny plant derived from the cross of (a); and (c) selectingprogeny that comprises the transgene, wherein the progeny is resistantagainst a coleopteran insect, lepidopteran insect, or a plant pathogenicnematode.

Seed can be produced from the plants of the present invention. Seedcontaining a polynucleotide molecule encoding one or more of theproteins of the present invention, whether homogyzous or heterozygousfor the particular transgenic allele, can be packaged for planting in afield, and a crop can be produced from the planted seed. The crop fromsuch plants can be harvested, and if seed of the harvested generationare the crop (such as soybean, rice, wheat, canola or corn and thelike), at least 50% of the harvested crop are seed containing thepolynucleotide molecule.

Commodity products (or biological samples) containing a plant or plantpart as set forth above that can be shown to contain a detectable amountof a polypeptide having the amino acid sequence of any of the proteinsof the present invention, or polynucleotides encoding any such protein.The detection of the polypeptide or the polynucleotide in the commodity(or biological sample) is determinative of the presence of the plant orplant part in the commodity (or biological sample), and all suchcommodity products in which the polypeptide is detectable to a level ofat least about (i) one part per million, (ii) or one nanogram per gramfresh weight of tissue, are within the scope of the present invention. Aplant cell of the present invention may be regenerated into arecombinant plant which can produce a plant part containing any of theproteins of the present invention. The plant part includes a leaf, astem a flower, a sepal, a fruit, a root, or a seed. Products producedfrom a recombinant plant or plant part contain a detectable amount ofany one of the proteins of the present invention, or polynucleotidesegments encoding such proteins. Such products include oil, meal, lintand seed of such recombinant plants. The detectable amount of theproteins and/or polynucleotides are useful as molecular markers fortracking and/or identifying the presence of seeds and plant tissues ofthe present invention as these are moved through commerce.

The proteins of the present invention originate from Bacillusthuringiensis species of bacteria, and as such, are likely to becharacterized as delta-endotoxins, and are typically produced from arecombinant polynucleotide. Such delta endotoxin proteins will have anamino acid sequence that exhibits at least from about 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% amino acidsequence identity to the amino acid sequence as set forth in any of thesequences shown in SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14,SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:34,SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:54,SEQ ID NO:58, and SEQ ID NO:60. Each such protein will preferablyinclude at least 50, or from about 50 to about 100, or from about 50 toabout 300 contiguous amino acids present in any full length proteinsequence set forth in the sequences referenced above, and the toxinproteins are preferably encoded by a polynucleotide segment thathybridizes under stringent conditions to the polynucleotide codingsequences as set forth in any of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ IDNO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ IDNO:57, SEQ ID NO:59, SEQ ID NO:61, and SEQ ID NO:63.

Compositions containing the proteins of the present invention areprovided in an agriculturally-acceptable carrier. The composition maycontain a recombinant Bacillus thuringiensis cell extract, cellsuspension, cell homogenate, cell lysate, cell supernatant, cellfiltrate, or cell pellet in which at least a pest inhibitory amount ofone or more of the proteins of the present invention are provided, andthe composition can be provided in the form of a powder, dust, pellet,granule, spray, emulsion, colloid, or solution. The composition may beprepared by desiccation, lyophilization, homogenization, extraction,filtration, centrifugation, sedimentation, or concentration of a cultureof recombinant Bacillus thuringiensis cells or spore ctystals containingone or more of the proteins of the present invention. The pesticidalcomposition preferably contains from about 1% to about 99% by weight ofone or more of the pesticidal proteins described herein.

The proteins of the present invention can be obtained in substantiallyconcentrated and/or purified form by a process which may include thesteps of i) culturing recombinant Bacillus thuringiensis cellscontaining one or more recombinant polynucleotide as set forth aboveunder conditions effective to produce the pesticidal protein, andobtaining the pesticidal polypeptide so produced. The polypeptide willpreferably contain the contiguous amino acid sequence as set forth inany of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ IDNO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:34, SEQ IDNO:38, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:54, SEQ IDNO:58, and SEQ ID NO:60.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO 1 represents a native Bt nucleotide sequence encoding a ET34protein.

SEQ ID NO 2 represents an amino acid sequence translation of SEQ ID NO1.

SEQ ID NO 3 represents an artificial sequence encoding a ET34 protein.

SEQ ID NO 4 represents an amino acid sequence translation of SEQ ID NO 3from nucleotide position 1 through nucleotide position 378.

SEQ ID NO 5 represents a nucleotide sequence encoding a P139 secretionsignal peptide (nucleotide position 1-75) fused in frame to a native Btnucleotide sequence encoding a ET34 protein (nucleotide position76-450).

SEQ ID NO 6 represents the amino acid sequence translation of SEQ ID NO5.

SEQ ID NO 7 represents a nucleotide sequence encoding a P139 secretionsignal peptide (nucleotide position 1-75) fused in frame to a syntheticnucleotide sequence encoding a ET34 protein (nucleotide position76-450).

SEQ ID NO 8 represents the amino acid sequence translation of SEQ ID NO7.

SEQ ID NO 9 represents the native Bt nucleotide sequence encoding aTIC1506 protein.

SEQ ID NO 10 represents the amino acid sequence translation of SEQ ID NO9.

SEQ ID NO 11 represents an artificial nucleotide sequence encoding aTIC1506 protein.

SEQ ID NO 12 represents the amino acid sequence translation of SEQ ID NO11.

SEQ ID NO 13 represents the native Bt nucleotide sequence encoding aTIC1501 protein.

SEQ ID NO 14 represents the amino acid sequence translation of SEQ ID NO13.

SEQ ID NO 15 represents an artificial nucleotide sequence encoding aTIC1501 protein.

SEQ ID NO 16 represents the amino acid sequence translation of SEQ ID NO15.

SEQ ID NO 17 represents the native Bt nucleotide sequence encoding aTIC1503 protein.

SEQ ID NO 18 represents the amino acid sequence translation of SEQ ID NO17.

SEQ ID NO 19 represents an artificial nucleotide sequence encoding aTIC1503 protein.

SEQ ID NO 20 represents the amino acid sequence translation of SEQ ID NO19.

SEQ ID NO 21 represents a native Bt nucleotide sequence encoding aTIC614 protein.

SEQ ID NO 22 represents an amino acid sequence translation of SEQ ID NO21.

SEQ ID NO 23 represents an artificial nucleotide sequence encoding aTIC614 protein.

SEQ ID NO 24 represents an amino acid sequence translation of SEQ ID NO23.

SEQ ID NO 25 represents a nucleotide sequence encoding a TIC615 protein.

SEQ ID NO 26 represents the amino acid sequence translation of SEQ ID NO25.

SEQ ID NO 27 represents an artificial nucleotide sequence encoding aTIC615 protein.

SEQ ID NO 28 represents the amino acid sequence translation of SEQ ID NO27.

SEQ ID NO 29 represents the native Bt nucleotide sequence encoding aTIC1277 protein.

SEQ ID NO 30 represents the amino acid sequence translation of SEQ ID NO29.

SEQ ID NO 31 represents an artificial nucleotide sequence encoding aTIC1277 protein.

SEQ ID NO 32 represents the amino acid sequence translation of SEQ ID NO31.

SEQ ID NO 33 represents the native Bt nucleotide sequence encoding a TICTIC1278 protein.

SEQ ID NO 34 represents the amino acid sequence translation of SEQ ID NO33.

SEQ ID NO 35 represents an artificial nucleotide sequence encoding a TICTIC1278 protein.

SEQ ID NO 36 represents the amino acid sequence translation of SEQ ID NO35.

SEQ ID NO 37 represents the native Bt nucleotide sequence encoding a TICTIC1310 protein.

SEQ ID NO 38 represents the amino acid sequence translation of SEQ ID NO37.

SEQ ID NO 39 represents an artificial nucleotide sequence encoding aTIC1310 protein.

SEQ ID NO 40 represents the amino acid sequence translation of SEQ ID NO39.

SEQ ID NO 41 represents the native Bt nucleotide sequence encoding a TICTIC1311protein.

SEQ ID NO 42 represents the amino acid sequence translation of SEQ ID NO41.

SEQ ID NO 43 represents an artificial nucleotide sequence encoding aTIC1311 protein.

SEQ ID NO 44 represents the amino acid sequence translation of SEQ ID NO43.

SEQ ID NO 45 represents the native Bt nucleotide sequence encoding aTIC1324 protein.

SEQ ID NO 46 represents the amino acid sequence translation of SEQ ID NO45.

SEQ ID NO 47 represents an artificial nucleotide sequence encoding aTIC1324 protein.

SEQ ID NO 48 represents the amino acid sequence translation of SEQ ID NO47.

SEQ ID NO 49 represents the native Bt nucleotide sequence encoding aTIC1407 protein.

SEQ ID NO 50 represents the amino acid sequence translation of SEQ ID NO49.

SEQ ID NO 51 represents an artificial nucleotide sequence encoding a TICTIC1407 protein.

SEQ ID NO 52 represents the amino acid sequence translation of SEQ ID NO51.

SEQ ID NO 53 represents the native Bt nucleotide sequence encoding a TICTIC1408 protein.

SEQ ID NO 54 represents the amino acid sequence translation of SEQ ID NO53.

SEQ ID NO 55 represents an artificial nucleotide sequence encoding aTIC1408 protein.

SEQ ID NO 56 represents the amino acid sequence translation of SEQ ID NO56.

SEQ ID NO 57 represents a native Bt nucleotide sequence encoding aTIC1308 protein.

SEQ ID NO 58 represents an amino acid sequence translation of SEQ ID NO57.

SEQ ID NO 59 represents a native Bt nucleotide sequence encoding aTIC1442 protein.

SEQ ID NO 60 represents an amino acid sequence translation of SEQ ID NO59.

SEQ ID NO 61 represents an artificial nucleotide sequence encoding aTIC1308 protein.

SEQ ID NO 62 represents an amino acid sequence translation of SEQ ID NO61.

SEQ ID NO 63 represents an artificial nucleotide sequence encoding aTIC1442 protein.

SEQ ID NO 64 represents an amino acid sequence translation of SEQ ID NO63.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to methods and compositions for pest control inplants, in particular nematode and/or insect control. In one aspect, theinvention relates to controlling, preventing or treating nematode and/orinsect infection in transgenic plants. The method comprises, in oneembodiment, generation of transgenic plants containing a recombinantconstruct and expression of such construct to impart such pestresistance to plants. The recombinant construct may comprise anucleotide sequence encoding one or more proteins, wherein the sequenceis operably linked to a heterologous promoter functional in a plantcell, and to cells transformed with the recombinant construct. Cellscomprising (meaning including but not limited to) the recombinantconstruct may be prokaryotic or eukaryotic. In particular, eukaryoticcells may be plant cells. Plants and seeds derived from such transformedplant cells are also contemplated. The transgenic plants or partsthereof of the present invention, in one embodiment, produce one or morepesticidal proteins derived from Bacillus thuringiensis bacterialstrains.

The present invention provides heterologous molecules that are expressedin the cytoplasm of the host cell, or if used in a eukaryotic cell suchas a plant cell, may also be directed into the plastid of the plant toprovide production of the toxic protein, and including, but not limitedto, nucleotide segments that encode polypeptides such as SEQ ID NO:2,SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQID NO:26, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:42, SEQ IDNO:46, SEQ ID NO:50, SEQ ID NO:54, SEQ ID NO:58, and SEQ ID NO:60 havingpesticidal activity. In certain embodiments, the polypeptide havingpesticidal activity may share at least about 45%, or at least about 50%,or at least about 51-79%, or at least 80%, or at least 85%, or at least90%, or at least 95%, or at least 98%, or at least 99%, or 100% sequenceidentity, to any one or more amino acid sequence(s) set forth in SEQ IDNO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, SEQ IDNO:22, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:38, SEQ IDNO:42, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:54, SEQ ID NO:58, or SEQ IDNO:60. The function of the encoded polypeptide may also be determined bymeasuring the efficacy of the presence of the transgene that encodes itin reducing nematode and/or insect infection, growth, reproduction, orsymptomology. For instance, a reduction in root galls, cysts, or wormnumber of 20% or more, 25% or more, 50% or more, 80% or more, or 95% ormore, in a transgenic plant comprising a heterologous nucleotideconstruct encoding any of the proteins of the present invention,relative to a control plant, for instance an otherwise isogenic plantnot comprising the heterologous molecule, under similar conditions,indicates the presence of a functional molecule.

In certain embodiments, a heterologous molecule provided by the presentinvention that is directed into the plastid of a plant to provideproduction of a toxin protein of the present invention may share atleast from about 60 to about 79%, or at least 80%, or at least 85%, orat least 90%, or at least 95%, or at least 98%, or at least 99%, or 100%sequence identity at the nucleotide level with one or more sequence(s)as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ IDNO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ IDNO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ IDNO:59, SEQ ID NO:61, and SEQ ID NO:63. Thus, in particular embodiments,the heterologous molecule may comprise a sequence encoding aheterologous chloroplast transit peptide.

Yet another aspect of the invention provides methods for production andfor use of one or more of the proteins of the present invention tocontrol nematode and/or insect infestation. Thus, methods for productionof a toxin, for instance in a plant cell, are provided. The toxin maythen be applied to soil prior to, during, or subsequent to planting of acrop, in order to control or reduce nematode infestation orsymptomatology of crop plants grown in that soil.

Unless otherwise noted, terms are to be understood according toconventional usage by those of ordinary skill in the relevant art.Definitions of common terms in molecular biology may also be found inRieger et al., Glossary of Genetics: Classical and Molecular, 5thedition, Springer-Verlag: New York, 1991; and Lewin, Genes V, OxfordUniversity Press: New York, 1994. The nomenclature for DNA bases as setforth at Title 37 of the United States Code of Federal Regulations, Part1, section 1.822.

As used herein, a “transgenic plant” is any plant in which one or more,or all, of the cells of the plant include a transgene. A transgene maybe integrated within a nuclear genome or organelle genome, or it may beextra-chromosomally replicating DNA. The term “transgene” means anucleic acid that is partly or entirely heterologous, foreign, to atransgenic microbe, plant, animal, or cell into which it is introduced.Cells that make up various cell and tissue types of plants include butare not limited to seed, root, leaf, shoot, flower, pollen and ovule.

“Recombinant DNA” is a polynucleotide having a genetically engineeredmodification introduced through combination of endogenous and/orexogenous molecules in a transcription unit, manipulation viamutagenesis, restriction enzymes, and the like or simply by insertingmultiple copies of a native transcription unit. Recombinant DNA maycomprise DNA segments obtained from different sources, or DNA segmentsobtained from the same source, but which have been manipulated to joinDNA segments which do not naturally exist in the joined form. Anisolated recombinant polynucleotide may exist, for example as a purifiedmolecule, or integrated into a genome, such as a plant cell, ororganelle genome or a microbe plasmid or genome. The polynucleotidecomprises linked regulatory molecules that cause transcription of an RNAin a plant cell.

As used herein, “percent identity” means the extent to which twooptimally aligned DNA or protein segments are invariant throughout awindow of alignment of components, for example nucleotide sequence oramino acid sequence. An “identity fraction” for aligned segments of atest sequence and a reference sequence is the number of identicalcomponents that are shared by sequences of the two aligned segmentsdivided by the total number of sequence components in the referencesegment over a window of alignment which is the smaller of the full testsequence or the full reference sequence. “Percent identity” (“%identity”) is the identity fraction times 100.

“Expression” means transcription of DNA to produce RNA. The resultingRNA may be without limitation mRNA encoding a protein, antisense RNA, ora double-stranded RNA for use in RNAi technology. Expression also mayrefer to translation of RNA, i.e. the production of encoded protein froman mRNA.

As used herein, “promoter” means regulatory DNA molecules forinitializing transcription. A “plant promoter” is a promoter capable ofinitiating transcription in plant cells whether or not its origin is aplant cell. For example it is well known that certain Agrobacteriumpromoters are functional in plant cells. Thus, plant promoters includepromoter DNA obtained from plants, plant viruses (in particular, doublestranded DNA viruses) and bacteria such as Agrobacterium andBradyrhizobium bacteria. Constitutive promoters generally providetranscription in most or all of the cells of a plant. In particular,promoters such as the FMV promoter (FMV, U.S. Pat. No. 6,051,753), theenhanced 35S promoter (E35S, U.S. Pat. No. 5,359,142), rice actinpromoter (U.S. Pat. No. 5,641,876), and various chimeric promoters (U.S.Pat. No. 6,660,911) are useful in the present invention. Examples ofpromoters under developmental control include promoters thatpreferentially initiate transcription in certain tissues, such asleaves, roots, or seeds. Such promoters are referred to as“tissue-preferred”. Promoters that initiate transcription only incertain tissues are referred to as “tissue specific.”

A number of root-specific or root-enhanced promoters or fragments ofsuch that provide enhanced expression in root tissues relative to otherplant tissues have been identified and are known in the art (e.g. U.S.Pat. Nos. 5,110,732, 5,837,848, 5,837,876; 5,633,363; 5,459,252;5,401,836; 7,196,247; 7,232,940; 7,119,254; and 7,078,589). Examplesinclude root-enhanced or root-specific promoters such as theCaMV-derived as-1 promoter or the wheat PDX1 promoter (U.S. Pat. No.5,023,179), the acid chitinase gene promoter (Samac et al., Plant Mol.Biol. 25:587-596 (1994); the root specific subdomains of the CaMV35Spromoter (Lam et al., Proc. Natl. Acad. Sci. (U.S.A.) 86:7890-7894(1989); the root-enhanced ORF13 promoter from Agrobacterium rhizogenes(Hansen et al., Mol. Gen. Genet. 254:337-343 (1997); the promoter forthe tobacco root-specific gene RB7 (U.S. Pat. No. 5,750,386); and theroot cell-specific promoters reported by Conkling et al. (Plant Physiol.93:1203-1211 (1990). Additional examples include RCc2 and RCc3,promoters that direct root-specific gene transcription in rice (Xu etal., Plant Mol. Biol. 27:237, 1995); soybean root-specific glutaminesynthetase promoter (Hire et al., Plant Mol. Biol. 20:207-218, 1992);root-specific control element in the GRP 1.8 gene of French bean (Kellerand Baumgartner, Plant Cell 3:1051-1061, 1991.); a root-specificpromoter of the mannopine synthase (MAS) gene of Agrobacteriumtumefaciens (Sanger et al., Plant Mol. Biol. 14:433-443, 1990); andfull-length cDNA clone encoding cytosolic glutamine synthetase (GS),which is expressed in roots and root nodules of soybean (Miao et al.,Plant Cell 3:11-22, 1991). See also Bogusz et al., Plant Cell 2:633-641,1990, where two root-specific promoters isolated from hemoglobin genesfrom the nitrogen-fixing non-legume Parasponia andersonii and therelated non-nitrogen-fixing non-legume Trema tomentosa are described.Leach and Aoyagi (1991) describe their analysis of the promoters of thehighly expressed rolC and rolD root-inducing genes of Agrobacteriumrhizogenes (see Plant Science (Limerick) 79:69-76). Additionalroot-preferred promoters include the VfENOD-GRP3 gene promoter (Kusteret al., Plant Mol. Biol. 29(4):759-772, 1995); and rolB promoter (Capanaet al., Plant Mol. Biol. 25:681-691, 1994). Examples of nematode-inducedpromoters include, for instance, the TobRB7 promoter (Opperman et al.,Science 263:221-223, 1994), and promoters described in U.S. Pat. Nos.6,262,344, and 7,193,136.

The term “resistance,” or “tolerance” when used in the context ofcomparing the effectiveness of a transgene in a transgenic plant, refersto the ability of the transgenic plant to maintain a desirable phenotypewhen exposed to nematode infestation pressures relative to the phenotypepresented by a nematode sensitive non-transgenic plant under similarconditions. The level of resistance can be determined by comparing thephysical characteristics of the transgenic plant to non-transgenicplants that either have or have not been exposed to nematode and/orinsect infection. Exemplary physical characteristics to observe includeplant height, an increase in population of plants that have ability tosurvive nematode or insect challenge (that is, plants that come incontact with a parasitic nematode or insect may have enhanced rootgrowth, enhanced fruit or grain yield, and decreased reproduction of thenematode or insect infesting the plant or crop, or a decrease in therate of increase if the pest population). The product of expression ofthe recombinant DNA may be directly toxic to the nematode (nematicidal)or insect (insecticidal), or may affect the mobility, host finding,feeding site establishment, fecundity or have other nematistatic and/orinsectic inhibitory effects.

“Transformed seed” is the seed which has been generated from thetransformed plant. A transformed plant contains transformed cells. Atransformed cell is a cell that has been altered by the introduction ofan exogenous DNA molecule or in the present invention comprises aheterologous DNA encoding one or more of the proteins of the presentinvention.

Pests intended to be within the scope of the present invention includethe “lepidopteran pest population” such as Spodoptera frugiperda,Spodoptera exigua, Mamestra configurata, Agrotis ipsilon, Trichoplusiani, Pseudoplusia includens, Anticarsia gemmatalis, Hypena scabra,Heliothis virescens, Agrotis subterranea, Pseudaletia unipuncta, Agrotisorthogonia, Ostrinia nubilalis, Amyelois transitella Crambuscaliginosellus, Herpetogramma licarsisalis, Homoeosoma electellum,Elasmopalpus lignosellu, Cydia pomonella, Endopiza viteana, Grapholitamolesta, Suleima helianthana, Plutella xylostella, Pectinophoragossypiella, Lymantria dispar, Blatta orientalis, Blatella asahinai,Blattella germanica, Supella longipalpa, Periplaneta americana,Periplaneta brunnea, Leucophaea maderae, Alabama argillacea, Archipsargyrospila, A. rosana, Chilo suppressalis, Cnaphalocrocis medinalis,Crambus caliginosellus, C. teterrellus, Diatraea grandiosella, D.saccharalis, Earias insulana, E. vittella, Helicoverpa armigera, H. zea,Heliothis virescens, Herpetogramma licarsisalis, Lobesia botrana,Pectinophora gossypiella, Phyllocnistis citrella, Pieris brassicae, P.rapae, Plutella xylostella, Spodoptera exigua, S. litura, S. frugiperda,and Tuta absoluta. The “coleopteran pest population” includes Anthonomusgrandis, Lissorhoptrus oryzophilu, Sitophilus granaries, Sitophilusoryzae, Hypera punctata, Sphenophorus maidis, Leptinotarsa decemlineata,Diabrotica virgifera virgifera, Diabrotica barberi, Diabroticaundecimpunctata howardi, Chaetocnema pulicaria, Phyllotreta cruciferae,Colaspis brunnea, Oulema melanopus, Zygogramma exclamationis, Epilachnavarivestis, Popillia japonica, Cyclocephala boreali, Cyclocephalaimmaculata, Rhizotrogus majalis, Phyllophaga crinita, Ligyrus gibbosus,Melanotus spp., Conoderus spp., Limonius spp., Agriotes spp., Cteniceraspp., and Aeolus spp., Eleodes spp. The “plant pathogenic nematodepopulation” includes plant parasitic species, for example, Heteroderaspecies, Globodera species, Meloidogyne species, Rotylenchulus species,Hoplolaimus species, Belonolaimus species, Pratylenchus species,Longidorus species, Paratrichodorus species, Ditylenchus species,Xiphinema species, Dolichodorus species, Helicotylenchus species,Radopholus species, Hirschmanniella species, Tylenchorhynchus species,and Trichodorus species, and the like, and specifically includesHeterodera glycines (soybean cyst nematode), Heterodera schachtii (beetcyst nematode), Heterodera avenae, Globodera rostochiensis, Globoderapailida, Pratylenchus zeae (a root knot nematode), Meloidogyne javanica,Pratylenchus brachyurus (a root knot nematode), Meloidogyne hapla, andMeloidogyne incognita.

The present invention provides recombinant DNA constructs comprising apolynucleotide that, when incorporated in a plant cell, imparts to theplant resistance to nematode and/or insect infection or plant diseasecaused by such infection (also referred to as infestation). Suchconstructs also typically comprise a promoter operatively linked to saidpolynucleotide to provide for expression in the plant cells. Otherconstruct components may include additional regulatory molecules, suchas 5′ leader regions or 3′ untranslated regions (such as polyadenylationsites), intron regions, and transit or signal peptides. Such recombinantDNA constructs can be assembled using methods known to those of ordinaryskill in the art.

Recombinant constructs prepared in accordance with the present inventionalso generally include a 3′ untranslated DNA region (UTR) that typicallycontains a polyadenylation sequence following the polynucleotide codingregion. Examples of useful 3′ UTRs include but are not limited to thosefrom the nopaline synthase gene of Agrobacterium tumefaciens (nos), agene encoding the small subunit of a ribulose-1,5-bisphosphatecarboxylase-oxygenase (rbcS), and the T7 transcript of Agrobacteriumtumefaciens.

Constructs and vectors may also include a transit peptide for targetingof a protein product, particularly to a chloroplast, leucoplast or otherplastid organelle, mitochondria, peroxisome, or vacuole or anextracellular location. For descriptions of the use of chloroplasttransit peptides, see U.S. Pat. No. 5,188,642 and U.S. Pat. No.5,728,925. Many chloroplast-localized proteins are expressed fromnuclear genes as precursors and are targeted to the chloroplast by achloroplast transit peptide (CTP). Examples of other such isolatedchloroplast proteins include, but are not limited to those associatedwith the small subunit (SSU) of ribulose-1,5,-bisphosphate carboxylase,ferredoxin, ferredoxin oxidoreductase, the light-harvesting complexprotein I and protein II, thioredoxin F, enolpyruvyl shikimate phosphatesynthase (EPSPS) and transit peptides described in U.S. Pat. No.7,193,133. It has been demonstrated in vivo and in vitro thatnon-chloroplast proteins may be targeted to the chloroplast by use ofprotein fusions with a heterologous CTP and that the CTP is sufficientto target a protein to the chloroplast. Incorporation of a suitablechloroplast transit peptide, such as, the Arabidopsis thaliana EPSPS CTP(CTP2, Klee et al., Mol. Gen. Genet. 210:437-442, 1987), and the Petuniahybrida EPSPS CTP (CTP4, della-Cioppa et al., Proc. Natl. Acad. Sci. USA83:6873-6877, 1986) has been show to target heterologous EPSPS proteinsequences to chloroplasts in transgenic plants. The production ofglyphosate tolerant plants by expression of a fusion protein comprisingan amino-terminal CTP with a glyphosate resistant EPSPS enzyme is wellknown by those skilled in the art, (U.S. Pat. No. 5,627,061, U.S. Pat.No. 5,633,435, U.S. Pat. No. 5,312,910, EP 0218571, EP 189707, EP508909, and EP 924299). Those skilled in the art will recognize thatvarious chimeric constructs can be made that utilize the functionalityof a CTP to import various pesticidal proteins of the present inventioninto the plant cell plastid.

Stable methods for plant transformation include virtually any method bywhich DNA can be introduced into a cell, such as by direct delivery ofDNA (for example, by PEG-mediated transformation of protoplasts, byelectroporation, by agitation with silicon carbide fibers, and byacceleration of DNA coated particles), by Agrobacterium-mediatedtransformation, by viral or other vectors. One preferred method of planttransformation is microprojectile bombardment, for example, asillustrated in U.S. Pat. No. 5,015,580 (soy), U.S. Pat. No. 5,550,318(maize), U.S. Pat. No. 5,538,880 (maize), U.S. Pat. No. 6,153,812(wheat), U.S. Pat. No. 6,160,208 (maize), U.S. Pat. No. 6,288,312 (rice)and U.S. Pat. No. 6,399,861 (maize), and U.S. Pat. No. 6,403,865(maize).

Detailed procedures for Agrobacterium-mediated transformation of plants,especially crop plants, include, for example, procedures disclosed inU.S. Pat. Nos. 5,004,863, 5,159,135, 5,518,908, 5,846,797, and 6,624,344(cotton); U.S. Pat. Nos. 5,416,011, 5,569,834, 5,824,877, 5,914,4516,384,301, and 7,002,058 (soy); U.S. Pat. Nos. 5,591,616 5,981,840, and7,060,876 (maize); U.S. Pat. Nos. 5,463,174 and 5,750,871 (Brassicaspecies, including rapeseed and canola), and in U. S. Patent ApplicationPublications 2004/0244075 (maize), 2004/0087030 (cotton) and2005/0005321 (soybean). Additional procedures for Agrobacterium-mediatedtransformation are disclosed in WO9506722 (maize). Similar methods havebeen reported for many plant species, both dicots and monocots,including, among others, peanut (Cheng et al., Plant Cell Rep., 15:653,1996); asparagus (Bytebier et al., Proc. Natl. Acad. Sci. U.S.A.,84:5345, 1987); barley (Wan and Lemaux, Plant Physiol., 104:37, 1994);rice (Toriyama et al., Bio/Technology, 6:10, 1988; Zhang et al., PlantCell Rep., 7:379, 1988; wheat (Vasil et al., Bio/Technology, 10:667,1992; Becker et al., Plant J., 5:299, 1994), alfalfa (Masoud et al.,Transgen. Res., 5:313, 1996); Brassica species (Radke et al., Plant CellRep., 11:499-505, 1992); and tomato (Sun et al., Plant Cell Physiol.,47:426-431, 2006). Transgenic plant cells and transgenic plants can alsobe obtained by transformation with other vectors, such as but notlimited to viral vectors (for example, tobacco etch virus (TEV), barleystripe mosaic virus (BSMV), and the viruses referenced in Edwardson andChristie, “The Potyvirus Group: Monograph No. 16”, 1991, Agric. Exp.Station, Univ. of Florida), plasmids, cosmids, YACs (yeast artificialchromosomes), BACs (bacterial artificial chromosomes) or any othersuitable cloning vector, when used with an appropriate transformationprotocol such as but not limited to bacterial infection (for example,with Agrobacterium as described above), binary bacterial artificialchromosome constructs, direct delivery of DNA (for example, viaPEG-mediated transformation, desiccation/inhibition-mediated DNA uptake,electroporation, agitation with silicon carbide fibers, andmicroprojectile bombardment). It would be clear to one of ordinary skillin the art that various transformation methodologies can be used andmodified for production of stable transgenic plants from any number ofplant species of interest. For example the construction of stablyinherited recombinant DNA constructs and mini-chromosomes can be used asvectors for the construction of transgenic plants (U.S. Pat. No.7,235,716).

Plants of the present invention include, but are not limited to, Acacia,alfalfa, aneth, apple, apricot, artichoke, arugula, asparagus, avocado,banana, barley, beans, beet, blackberry, blueberry, broccoli, brusselssprouts, cabbage, canola, cantaloupe, carrot, cassava, cauliflower,celery, cherry, cilantro, citrus, clementine, coffee, corn, cotton,cucumber, Douglas fir, eggplant, endive, escarole, eucalyptus, fennel,figs, forest trees, gourd, grape, grapefruit, honey dew, jicama,kiwifruit, lettuce, leeks, lemon, lime, loblolly pine, mango, melon,mushroom, nut, oat, okra, onion, orange, an ornamental plant, papaya,parsley, pea, peach, peanut, pear, pepper, persimmon, pine, pineapple,plantain, plum, pomegranate, poplar, potato, pumpkin, quince, radiatapine, radicchio, radish, rapeseed, raspberry, rice, rye, sorghum,Southern pine, soybean, spinach, squash, strawberry, sugarbeet,sugarcane, sunflower, sweet potato, sweetgum, tangerine, tea, tobacco,tomato, turf, a vine, watermelon, wheat, yams, and zucchini. Crop plantsare defined as plants which are cultivated to produce one or morecommercial products. Examples of such crops or crop plants include butare not limited to soybean, canola, rape, cotton (cottonseeds), peanut,sunflower, pigeon pea, chickpea, and the like, and grains such as corn,wheat, rice, oat, millet, and rye, and the like. Rape, rapeseed andcanola are used synonymously in the present disclosure.

Transformation methods to provide transgenic plant cells and transgenicplants containing stably integrated recombinant DNA are preferablypracticed in tissue culture on media and in a controlled environment.Recipient cell targets include but are not limited to meristem cells,callus, immature embryos or parts of embryos, gametic cells such asmicrospores, pollen, sperm, and egg cells. Any cell from which a fertileplant can be regenerated is contemplated as a useful recipient cell forpractice of the invention. Callus can be initiated from various tissuesources, including, but not limited to, immature embryos or parts ofembryos, seedling apical meristems, microspores, and the like. Thosecells which are capable of proliferating as callus can serve asrecipient cells for genetic transformation. Practical transformationmethods and materials for making transgenic plants of this invention(for example, various media and recipient target cells, transformationof immature embryos, and subsequent regeneration of fertile transgenicplants) are disclosed, for example, in U.S. Pat. Nos. 6,194,636 and6,232,526 and U. S. Patent Application Publication 2004/0216189.

In general transformation practice, DNA is introduced into only a smallpercentage of target cells in any one transformation experiment. Markergenes are generally used to provide an efficient system foridentification of those cells that are transformed by a transgenic DNAconstruct. Preferred marker genes provide selective markers which conferresistance to a selective agent, such as an antibiotic or herbicide. Anyof the antibiotics or herbicides to which a plant cell may be resistantcan be a useful agent for selection. Potentially transformed cells areexposed to the selective agent. In the population of surviving cellswill be those cells where, generally, the resistance-conferring gene isexpressed at sufficient levels to permit cell survival in the presenceof the selective agent. Cells can be tested further to confirmintegration of the recombinant DNA. Commonly used selective marker genesinclude those conferring resistance to antibiotics such as kanamycin orparomomycin (val), hygromycin B (aph IV), gentamycin (aac3 and aacC4)and glufosinate (bar or pat), glyphosate (EPSPS), and dicamba (dicambamonooxygenase). Examples of useful selective marker genes and selectionagents are illustrated in U.S. Pat. Nos. 5,550,318, 5,633,435,5,780,708, and 6,118,047. Screenable markers or reporters, such asmarkers that provide an ability to visually identify transformants canalso be employed. Non-limiting examples of useful screenable markersinclude, for example, a gene expressing a protein that produces adetectable color by acting on a chromogenic substrate (for example,beta-glucuronidase, GUS, uidA, or luciferase, luc) or that itself isdetectable, such as green fluorescent protein (GFP, gfp) or animmunogenic molecule. Those of skill in the art will recognize that manyother useful markers or reporters are available for use.

The recombinant DNA constructs of the invention can be stacked withother recombinant DNA for imparting additional agronomic traits (such asin the case of transformed plants, traits including but not limited toherbicide resistance, insect resistance, cold germination tolerance,water deficit tolerance, enhanced yield, enhanced quality, fungal,viral, and bacterial disease resistance) for example, by expressingother transgenes. The recombinant DNA constructs of the presentinvention can also be transformed into plant varieties that carrynatural pest or pathogen resistance genes to enhance the efficacy of theresistance phenotype. Constructs for coordinated decrease and/orincrease of gene expression are disclosed in U.S. Patent ApplicationPublication 2004/0126845 A1. Seeds of transgenic, fertile plants can beharvested and used to grow progeny generations, including hybridgenerations, of transgenic plants of this invention that include therecombinant DNA construct in their genome. Thus, in addition to directtransformation of a plant with a recombinant DNA construct of thisinvention, transgenic plants of the invention can be prepared bycrossing a first plant having the recombinant DNA with a second plantlacking the construct. For example, the recombinant DNA can beintroduced into a plant line that is amenable to transformation toproduce a transgenic plant, which can be crossed with a second plantline to introgress the recombinant DNA into the resulting progeny. Atransgenic plant of the invention can be crossed with a plant linehaving other recombinant DNA or naturally occurring genetic regions thatconfers one or more additional trait(s) (such as, but not limited to,herbicide resistance, pest or disease resistance, environmental stressresistance, modified nutrient content, and yield improvement) to produceprogeny plants having recombinant DNA that confers both the desiredtarget sequence expression behavior and the additional trait(s).Typically, in such breeding for combining traits the transgenic plantdonating the additional trait is a male line and the transgenic plantcarrying the base traits is the female line. The progeny of this crosssegregate such that some of the plant will carry the DNA for bothparental traits and some will carry DNA for one parental trait; suchplants can be identified by markers associated with parental recombinantDNA. Progeny plants carrying DNA for both parental traits can be crossedback into the female parent line multiple times, for example, usually 6to 8 generations, to produce a progeny plant with substantially the samegenotype as one original transgenic parental line but for therecombinant DNA of the other transgenic parental line.

Other proteins and toxic agents can be used together with one or moreproteins of the present invention to control plant pathogenic nematodeand/or insect infestation and to reduce the likelihood of development ofresistance to any single method of control. Such other proteins andtoxic agents include but are not limited to, as applicable to eithernematode or insect control, methylketone synthase, dsRNA expressed inthe cell and targeting for suppression one or more essential,housekeeping, reproductive or developmental gene, other proteins thatare known in the art to be toxic to plant pathogenic nematodes orinsects such as Cry and VIP proteins(lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/index.html” on the worldwide web, which is properly referenced as Crickmore et. al. (2010)“Bacillus thuringiensis toxin nomenclature”), as well as chemicalnematicides used in seed treatments or soil drenches. Topically applieddsRNA methods are also known in the art that can be applied to a plantexpressing one or more of the proteins of the present invention. Suchtopical applications can be effective in causing a systemic effect inthe plant that result in nematode or insect control by applying to theplant a dsRNA molecule that targets for regulation a gene in the plantinvolved in such resistance. All such combinations are within the scopeof the present invention.

The transgenic plant, plant part, seed or progeny plants of the presentinvention can be processed into products useful in commerce. Theseproducts, commodity products, include but are not limited to meal,flour, oil, hay, starch, juice, protein extract, and fiber.

The proteins of the present invention have been identified using avariety of methods. One method has been to identify previously known Btproteins that exhibit a mass less than about 40 kDa, or less than about35 kDa, or less than about 30 kDa, or less than about 28 kDa, or lessthan about 25 kDa, or less than about 20 kDa, or less than about 15 kDa.Such proteins include but are not limited to the smaller component ofmost known binary Bt toxins, such as Cry34/35 (PS149B1), TIC100/101,ET33/34, ET80/76, and the like. Other proteins known in the art includeTIC901, TIC1201, TIC407, TIC417, TIC431, ET70, VIP proteins such asVIP3Aa and the like, which are all generally small toxin proteins thatare known to exhibit insecticidal activity. The inventors herein haveidentified that such smaller toxin molecules, when provided in the dietof a C. elegans nematode, exhibit various levels of inhibitory effects.Surprisingly, it has also been observed that the nematidical activity ofthese proteins can be imparted through the diet of a cyst nematode moreeffectively by truncating the proteins to smaller sizes, whethertruncated at the C terminus, the N terminus, or both. Truncated versionstypically exhibit a mass of from about 14 to about 28-30 kDa, andexhibit improved bioactivity likely because the ability of the cystnematode to uptake proteins larger than about 30 kDa is limited (Urwinet al. ((1997) Plant J. 12:455) and Bockenhoff & Grundler ((1994)Parasitology 109:249). TIC1501 (about 27 kDa), TIC1503 (about 34 kDa),and TIC1506 (about 36 kDa) represent various fragments of the TIC1201protein, 1201 being previously shown to exhibit coleopteran toxiceffects. Surprisingly, the truncated versions less than 36 kDa exhibitsignificant nematicidal effects.

Proteins of the present invention have also been identified de novo, andthese include the proteins listed herein as TIC614, TIC615, TIC1277,TIC1278, TIC1308, TIC1310, TIC1311, TIC1324, TIC1407, TIC1408, andTIC1442. Such proteins were identified by various methods, whether beingdirectly amplified from various Bt strain genomes, or identified by highthroughput sequence analysis of various Bt genomes. In either case,genomic DNA segments are obtained and analyzed using bioinformatictechniques that result in the identification of all or portions of openreading frames encoding protein segments. The resulting protein segmentsare then characterized versus all known protein sequences in the art,and to the extent that there is any similarity to a toxin molecule, thecomplete sequence of the open reading frame encoding the protein isobtained. Proteins that are identified that exhibit a mass of less thanabout 40 kDa, or preferably less than about 30 kDa are then evaluated ina C. elegans assay to determine if any effects are observed relative toC. elegans survival. Toxins exhibiting nematicidal properties are thenevaluated for other pesticidal properties, particularly insecticidalactivity. Surprisingly, the above referenced proteins all exhibitednematicidal activity, and some exhibited insecticidal activity asreported in the examples below.

EXAMPLES

The following examples are illustrative of the invention, which may beembodied in various forms and are not to be interpreted as limiting thescope or content of the disclosure in any way.

Example 1 DNA Molecules Encoding Bt Toxin Proteins

Toxin ET34 (SEQ ID NO:1) has been previously described (U.S. Pat. No.6,063,756). A secretion signal from the gene P139 (First 75 nucleotideof SEQ ID NO:1 from the WIPO Publication Number WO9408010) was operablylinked to the 5′ end of the ET34 (SEQ ID NO:5) to enable its secretionoutside the plasma membrane to avoid potential toxicity to the plantcell and to allow easy access of the protein to the pest.

TIC1506 (SEQ ID NO:9), TIC1501 (SEQ ID NO:13), and TIC 1503 (SEQ IDNO:17) are Bt nucleotide of various fragments of TIC1201 which is 364amino acids long as set forth in SEQ ID NO 6 of US Patent ApplicationPublication Number US2006-0191034 A1. TIC1506 is 321 amino acids longwithout the putative N terminal signal peptide of TIC1201 and containsamino acid 44 to 364 of TIC1201 with a methionine residue substitutedfor the native alanine residue at amino acid position 44. TIC1501 is 227amino acids long without the putative N terminal signal peptide and aportion of the C terminal of TIC1201 and contains amino acids 44 to 270of TIC1201 with a methionine residue substituted for the native alanineresidue at position 44. TIC1503 is 301 amino acids long without theputative N terminal signal peptide and a portion of the C terminal ofTIC1201 and contains amino acids 44 to 344 of TIC1201 with a methionineresidue substituted for the native alanine residue at position 44.

Proteins exhibiting pesticidal properties have been identified invarious Bt strains. Open reading frames encoding the amino acidsequences, TIC614 (SEQ ID NO: 22), TIC615 (SEQ ID NO: 26), TIC1277 (SEQID NO: 30), TIC1278 (SEQ ID NO: 34), TIC1310 (SEQ ID NO: 38), TIC1311(SEQ ID NO: 42), TIC1324 (SEQ ID NO: 46), TIC1407(SEQ ID NO: 50), andTIC1408 (SEQ ID NO: 54), exhibiting various degrees of homology topreviously known Bt toxin segments were identified. Complete forward andreverse sequence analysis of such open reading frames resulted in theidentification of deduced amino acid compositions that exhibit the sizeand potential for pesticidal (nematicidal and/or insecticidal) activity.

Proteins exhibiting pesticidal properties have been identified invarious Bt strains. Open reading frames encoding the amino acidsequences, TIC1308 (SEQ ID NO: 58) and TIC1442 (SEQ ID NO: 60),exhibiting various degrees of homology to previously known Bt toxinsegments were identified. Complete forward and reverse sequence analysisof such open reading frames resulted in the identification of deducedamino acid compositions that exhibit the size and potential forpesticidal (nematicidal and/or insecticidal) activity.

Example 2 Expression of Pesticidal Polypeptides from Polynucleotides

Open reading frames of ET34 (SEQ ID NO: 1), P139-ET34 (SEQ ID NO: 5),TIC1506 (SEQ ID NO:9), TIC1501 (SEQ ID NO:13), TIC 1503 (SEQ ID NO:17),TIC614 (SEQ ID NO:21), TIC615 (SEQ ID NO:25), TIC1277 (SEQ ID NO:29),TIC1278 (SEQ ID NO:33), TIC1310 (SEQ ID NO:37), TIC1311 (SEQ ID NO:41),TIC1324 (SEQ ID NO:45), TIC1407 (SEQ ID NO:49), TIC1408 (SEQ ID NO:53),TIC1308 (SEQ ID NO: 57), and TIC1442 (SEQ ID NO: 59) encoding thededuced amino acid compositions exemplified in Example 1 were clonedinto a Bt/E. coli shuttle plasmid enabling the expression of the deducedamino acid composition in either an acrystalliferous Bt strain or in anE. coli bacterium. Recombinant plasmids were transformed into anacrystalliferous Bt expression host after confirming the DNA sequence ofthe polynucleotide encoding the polypeptide. The gene of interest wascloned downstream of either a Bacillus vegetative stage or sporulationstage specific promoter to allow the protein to be expressedrespectively during vegetative growth or during sporulation of therecombinant Bt strain. Conditions for vegetative expression of theprotein included growing the cells for 24-48 hrs in Terrific Brothmedium at 25-28° C. Crystal formation is one characteristic of certainBt toxin proteins. Proteins that were confirmed to produce crystals whenexpressed in the acrystalliferous Bt strain were further evaluated.Certain proteins accumulated in the cells and/or were secreted into theculture medium. Both the cell pellets and the culture were analyzed bySDS-PAGE for expression of the expected protein. Conditions forexpression from a sporulation specific promoter included growing thecells for 96 hrs at 25-28° C. in C2 medium. Protein crystals were formedduring sporulation and released from lysed cells as sporulation wascompleted. Spores and crystals were collected by centrifugation at4000×g for 20 minutes, resuspended in wash buffer (10 mM Tris, 0.1 mMEDTA and 0.005% Triton X100 pH 6.8) and collected again bycentrifugation. The spore-crystals pellets were then resuspended in1/10^(th) of the original culture volume. The 10× concentratedspore-crystal preparation were analyzed by SDS-PAGE the presence of theexpected protein.

Example 3 Nematode and Insect Bioassays

C. elegans feeding screens have been successfully used to identify plantpathogenic nematode-active toxins, for example for SCN and RKN from theorder Tylenchida (Wei et al., 2003, PNAS, USA, 100: 2760). The proteinsof the present invention were expressed and provided in the diet of a C.elegans nematode, essentially following the method of Wei et al.Efficacy was scored on a scale of 1-3, where a score of 1 representsnormal health and reproduction of C. elegans, and a score of 3represents no reproduction or poor health of C. elegans. Toxins ET34(SEQ ID NO: 2), TIC1501 (SEQ ID NO: 14), TIC1503 (SEQ ID NO: 18), TIC614(SEQ ID NO:22), TIC615 (SEQ ID NO:26), TIC1277 (SEQ ID NO:30), TIC1278(SEQ ID NO:34), TIC1310 (SEQ ID NO:38), TIC1311 (SEQ ID NO:42), TIC1324(SEQ ID NO:46), and TIC1407(SEQ ID NO:50), each exhibited a score of 3where as TIC1408 (SEQ ID NO:54) exhibited a score of 2.75. ProteinsTIC1308 (SEQ ID NO: 58) and TIC1442 (SEQ ID NO: 60), each exhibited ascore of 1. Proteins were expressed from a vegetative specific promoterand fed to insects by applying sporulated bacterial cells or culturesupernatant to artificial insect diet. For polypeptides expressed from asporulation specific promoter a 10-20× spore-crystal preparation withabout 500-4000 ppm protein was applied to the insect diet. Stunting ormortality was observed on one or more of these insects: CEW (Corn EarWorm); SCR (Southern corn Root worm); WCR (western corn root worm); ECB(European corn borer); WTPB (Western tarnished plant bug); TPB(Tarnished plant bug); FAW (Fall army worm); CPB (Colorado potatobeetle). TIC1277 and TIC1311 were found to cause significant stunting ofECB and TIC1310 was found to cause significant stunting of WTPB andsignificant stunting and mortality of CPB.

Example 4 Transformed Plants

Nucleotide segments encoding TIC1506 (SEQ ID NO:11), TIC1503(SEQ ID NO:19), TIC614 (SEQ ID NO:3), TIC1324 (SEQ ID NO:27), TIC1407(SEQ IDNO:31), TIC1408 (SEQ ID NO:35), are codon-optimized for plant expressionand operably linked to one or more plant functional promoters andintroduced into plant cells. Recombinant plants are regenerated fromsuch transformed plant cells, and the regenerated plants are evaluatedfor resistance to pest infestation, such as insect tolerance and/orplant pathogenic nematode tolerance.

Nucleotide segments encoding ET34 (SEQ ID NO:3), ET34+P139 secretionsignal (SEQ ID NO:7), TIC1501 (SEQ ID NO:15), TIC615 (SEQ ID NO:7),TIC1277 (SEQ ID NO:11), TIC1278 (SEQ ID NO:15), TIC1310 (SEQ ID NO:19),TIC1311 (SEQ ID NO:23), TIC1308 (SEQ ID NO: 61), and TIC1442 (SEQ ID NO:63) were codon-optimized for plant expression and operably linked to oneor more plant functional promoters and introduced into plant cells.Recombinant plants were regenerated from such transformed plant cells,and the regenerated plants were evaluated for resistance to pestinfestation, such as insect tolerance and/or plant pathogenic nematodetolerance.

Example 5 Transformation of Soybean

This example describes a method of producing transgenic soybean plantsand transgenic plant parts such as seeds. Other methods are known in theart of plant cell transformation that can be applied to transform plantcells and regenerate transgenic plants using the recombinant constructsof the invention. The methods of obtaining transgenic soybean plants andseeds are used as essentially disclosed in US Patent ApplicationPublication Number US2009-0138985A1. Briefly, Agrobacterium containing aconstruct of Example 5 are grown in Luria Burtani (LB) media containingspectinomycin at about 28° C. for over night. The bacterial culture iscentrifuged, pellet washed, and resuspended in INO medium forinoculating wet or dry mature embryos explants. The explants are mixedwith the Agrobacterium cell suspensions and briefly exposed tosonication energy from a standard laboratory water bath cleaningsonicator. The explants are drained of any liquid and transferred tocontainers containing filter paper moistened with INO media andco-cultured in a lighted chamber at about 16 hours of light (5 uE) atabout 23° to 28 C.° for 1 to 5 days. After co-culture, the explants areplaced directly onto regeneration media containing a selective agentsuch as spectinomycin from about 7 to about 42 days. The cultures aresubsequently transferred to a media suitable for the recovery oftransformed plantlets. Spectinomycin resistant shoots that have greenbuds or leaves are considered transformed and placed in soil or on asoil substitute for rooting in the presence or absence of the selectiveagent. Progeny transgenic plants and seed are selected that provide pestresistance, especially nematode resistance.

Example 6 Testing of Transgenic Plant for Soybean Cyst Nematode (SCN)Resistance

An SCN pot assay was used to evaluate the resistance of transgenicsoybean plants comprising one or more of the polynucleotide sequences ofSEQ ID NOs: 3, 7, and 15 to infection by and reproduction of the SCN(Heterodera glycines) on roots. Three or four inch diameter square potswere filled with clean sand and watered thoroughly. Transgenic andcontrol soybean seeds, or alternatively any rooted plant parts, wereplanted one per pot in the center of the pot and watered well to removeair pockets. The pots were incubated in the greenhouse or growth chamberat 20° C. to 30° C. until the plants reached a suitable age forinoculation. Soybeans started from seed were typically inoculated 2-3weeks after planting, while transplants were inoculated 1-3 days afterplanting. The test inoculum consisted of eggs from ripe H. glycinescysts collected from the soil and roots of infested soybean plants. An80 micron mesh sieve was used to collect the cysts, which were thencrushed in a Tenbroeck glass tissue homogenizer to release the eggs. Theeggs were further purified by sieving and centrifugation over 40 percentsucrose solution at 4000 RPM for 5 minutes. Inoculum for an experimentconsisted of water containing 500 vermiform eggs per mL. Five mL of theegg suspension was applied over the surface of the sand containing thetest plants and the eggs were lightly watered in. The test plants werethen returned to the greenhouse or growth chamber and incubated for 3-4weeks to allow for root infection and cyst formation. The roots werethen harvested. The severity of nematode infection was measured bycounting the number of nematode cysts adhering to the root system.

Transgenic soybean plants comprising SEQ ID NO: 3 were tested in sixdifferent constructs, where in each construct SEQ ID NO: 3 was operablylinked to a different promoter. Transgenic soybean plants comprising SEQID NO: 7 were tested in two different constructs, each construct havinga different promoter. Transgenic soybean plants comprising SEQ ID NO: 15were tested expressed from one construct.

Table 1 reports data illustrating plants from multiple events permultiple constructs that were evaluated for and determined to havesignificant cyst reduction against SCN when compared to theuntransformed soybean cultivar. The number of plant roots tested wasabout equally distributed among the number of events tested.

TABLE 1 Severity of Soybean Cyst Nematode cyst infection on soybeanplant roots Number Number of SEQ of plant Result (compared to Protein IDConstruct Events roots control non-transgenic Name NO: Name testedtested plants) ET34 3 128213 14 177 1 of 14 events showed 35.4% cystreduction ET34 3 126168 15 389 7 of 15 events showed more than 46.7%cyst reduction ET34 3 126626 15 191 0 of 15 events showed significantcyst reduction ET34 3 126630 16 204 1 of 16 events showed 42.0% cystreduction ET34 3 127056 15 191 3 of 15 events showed more than 41.5%cyst reduction ET34 3 128296 14 168 2 of 14 events showed more than 33%cyst reduction P139 + 7 126169 15 176 0 of 15 events showed ET34 thesignificant cyst reduction P139 + 7 126628 16 192 0 of 16 events showedET34 the significant cyst reduction TIC1501 15 133535 5 18 3 of 5 eventsshowed more than 45% cyst reduction

Example 7 Testing of Transgenic Arabidopsis Plant for Beet Cyst Nematode(BCN) Resistance

Transgenic Arabidopsis seeds and plants comprising one or more of thepolynucleotide sequences of SEQ ID NOs 3, 15, 27, 31, 35, 39, 43, 61,and 63 were produced by the method of Clough et al., 1998 (Plant J.16:735-743) and tested for Beet Cyst Nematode (BCN) resistance by themethod of Sijmons et al., 1991 (Plant J. 1: 245-254) and Vaghchhipawalaet al., 2004 (Genome 47: 404-13).

Arabidopsis (variety Columbia-0) seeds were surface sterilized andrinsed with sterile water and plated on B5 medium. Plates were incubatedat 23-25° C. with a 16 hour light/8 hour dark cycle for 7-10 days. BCNeggs were placed on the sterile filter paper and hatched in 5 mM ZnSO4solution for 5-7 days at 25° C. J2 stage juvenile nematodes werecollected, rinsed in sterile water, and treated with 0.5% chlorhexidinediacetate for 10-15 minutes. Treated juvenile J2 nematodes werecollected and rinsed twice in sterile water and stored in sterile waterfor infestation purposes.

For the infestation assay, about 10-15 Arabidopsis seeds were sprinkledon steamed sand in a pot and covered with a clear plastic dome. Severalsuch dome/flat combos were placed in a flat and then covered with ablack tray and transferred to a cold room for vernalization. On day 4,the flat was taken out of the cold room, the black tray is removed, andthe flat was placed in a growth chamber for acclimating seeds at 26° C.,70% humidity, 140-180 μE light, 12 hours day length. The pots werewatered and fertilized as needed. Three weeks after planting, theArabidopsis plants were inoculated with 3,000 BCN eggs. About 35 daysafter inoculation the plants were harvested and cysts extracted bywashing the plant's roots in a bucket of water and filtering the waterthrough a 16 mesh sieve on top of a 50 mesh sieve. The cysts werecollected off the top of the 50 mesh sieve and counted. Plants withlesser number of cysts compared to non-transgenic or transgenic controlwere considered resistant to BCN.

Table 2 reports data illustrating plants from multiple events permultiple constructs that were evaluated for and determined to havelesser number of BCN cysts compared to the untransformed Arabidopsisparental background. The number of plant roots tested was nearly equallydistributed among the number of events tested.

TABLE 2 Severity of Beet Cyst Nematode cyst infection on Arabidopsisthaliana plant roots Number Resulting number Number of of events havinga SEQ of plant lower mean cyst count Protein ID Construct Events rootscompared to non- Name NO: Name tested tested transgenic control ET34 3140057 6 72 3 TIC1501 15 140056 6 72 3 TIC615 27 139822 6 72 4 TIC127731 142259 6 71 2 TIC1278 35 141647 6 72 4 TIC1310 39 142255 6 72 1TIC1311 43 141644 6 71 6 TIC1308 61 141250 6 70 2 TIC1422 63 141205 6 722

Various patent and non-patent publications are cited herein, thedisclosures of each of which are incorporated herein by reference intheir entireties. Documents cited herein as being available from theWorld Wide Web at certain internet addresses are also incorporatedherein by reference in their entireties.

As various modifications could be made in the compositions and methodsherein described and illustrated without departing from the scope of theinvention, it is intended that all matter contained in the foregoingdescription or shown in the accompanying drawings shall be interpretedas illustrative rather than limiting. Thus, the breadth and scope of thepresent invention should not be limited by any of the above-describedexemplary embodiments, but should be defined only in accordance with thefollowing claims appended hereto and their equivalents.

1-9. (canceled)
 10. A DNA construct comprising a polynucleotide operablylinked to a heterologous promoter, wherein said polynucleotide encodes apesticidal polypeptide that comprises an amino acid sequence at least95% identical to SEQ ID NO:42.
 11. The DNA construct of claim 10,wherein said polynucleotide encodes a pesticidal polypeptide thatcomprises an amino acid sequence at least 99% identical to SEQ ID NO:42.12. The DNA construct of claim 10, wherein said polynucleotide encodes apesticidal polypeptide that comprises the amino acid sequence as setforth in SEQ ID NO:42.
 13. The DNA construct of claim 10, wherein saidpolynucleotide is codon-optimized for expression in a plant.
 14. The DNAconstruct of claim 13, wherein said polynucleotide comprises the nucleicacid sequence as set forth in SEQ ID NO:43.
 15. A host cell comprising aDNA construct that comprises a polynucleotide operably linked to aheterologous promoter, wherein said polynucleotide encodes a pesticidalpolypeptide that comprises an amino acid sequence at least 95% identicalto SEQ NO:42.
 16. The host cell of claim 15, wherein said polynucleotideencodes a pesticidal polypeptide that comprises an amino acid sequenceat least 99% identical to SEQ ID NO:42.
 17. The host cell of claim 15,wherein said polynucleotide encodes a pesticidal polypeptide thatcomprises the amino acid sequence as set forth in SEQ ID NO:42.
 18. Thehost cell of claim 15, wherein said host cell is a bacterial cell or aplant cell.
 19. The host cell of claim 18, wherein said bacterial cellis selected from the group consisting of an Agrobacterium, a Bacillus,an Escherichia, a Salmonella, a Pseudomonas, and a Rhizohium cell, andwherein said plant cell is selected from the group consisting of aalfalfa, banana, barley, bean, broccoli, cabbage, canola, carrot,cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus,coconut, coffee, corn, clover, cotton, a cucurbit, cucumber, Douglasfir, eggplant, eucalyptus, flax, garlic, grape, hops, leek, lettuce,Loblolly pine, millets, melons, nut, oat, olive, onion, ornamental,palm, pasture grass, pea, peanut, pepper, pigeonpea, pine, potato,poplar, pumpkin, Radiata pine, radish, rapeseed, rice, rootstocks, rye,safflower, shrub, sorghum, Southern pine, soybean, spinach, squash,strawberry, sugar beet, sugarcane, sunflower, sweet corn, sweet gum,sweet potato, switchgrass, tea, tobacco, tomato, triticale, turf grass,watermelon, and a wheat plant cell.
 20. A plant, or part thereof,comprising a polynucleotide encoding a pesticidal polypeptide thatcomprises an amino acid sequence at least 95% identical to SEQ ID NO:42.21. The plant, or part thereof, of claim 20, wherein said polynucleotidecomprises the nucleic acid sequence as set forth in SEQ ID NO:43. 22.The plant, or part thereof, of claim 20, wherein said plant is electedfrom the group consisting of a alfalfa, banana, barley, bean, broccoli,cabbage, canola, carrot, cassava, castor, cauliflower, celery, chickpea,Chinese cabbage, citrus, coconut, coffee, corn, clover, cotton, acucurbit, cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic,grape, hops, leek, lettuce, Loblolly pine, millets, melons, nut, oat,olive, onion, ornamental, palm, pasture grass, pea, peanut, pepper,pigeonpea, pine, potato, poplar, pumpkin, Radiata pine, radish,rapeseed, rice, rootstocks, rye, safflower, shrub, sorghum, Southernpine, soybean, spinach, squash, strawberry, sugar beet, sugarcane,sunflower, sweet corn, sweet gum, sweet potato, switchgrass, tea,tobacco, tomato, triticale, turf grass, watermelon, and a wheat plant,and wherein said part is selected from the group consisting of a leaf, astem a flower, a sepal, a fruit, a root, or a seed.
 23. A method ofcontrolling a pest infection of a plant, said method comprisingproviding in a diet of said pest a plant, or part thereof, said plant orpart comprising a polynucleotide encoding a pesticidal polypeptide thatcomprises an amino acid sequence at least 95% identical to SEQ ID NO:42.24. The method of claim 23, wherein said pest is an insect or anematode.
 25. The method of claim 24, wherein said insect is an insectfrom the insect order selected from the group consisting of Coleoptera,Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera,Orthoptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera,and Trichoptera, and wherein said nematode is selected from the groupconsisting of Acontylus, Anguina, Aorolaimus, Aphasmatylenchus,Aphelenchoides, Aphelenchus, Atalodera, Atylenchus, BakernemaBelonolaimus, Brachydorus, Bursaphelenchus, Cacopaurus, Caloosia,Carphadorus, Criconema, Criconemella, Cryphodera, Ditylenchus,Dolichodorus, Eutylenchus, Globodera, Gracilacus, Helicotylenchus,Hemicriconemoides, Hemicycliophora, Heterodera, Hirschmanniella,Histotylenchus, Hoplolaiinus, Hoplotylus, Longidorus, liacrotrophurus,Meloidodera, Meloidogyne, Merlinius, Morulaimus, Nacobbus, Nothanguina,Nothotylenchus, Paralongidorus, Paratrichodorus, Paratrophurus,Paratylenchus, Peltamigratus, Pratylenchoides, Pratylenchus, Psilenchus,Radopholoides, Radopholus, Rhadinaphelenchus, Rototylenchus,Rotylenchoides, Rotylenchus, Sarisodera, Scutellonema, Sphaeronema,Subanguina, Telotylenchoides, Telotylenchus, Trichotylenchus,Trophonerna, Trophotylenculus, Trophurus, Tylenchorhynchus, Tylenchulus,Tylenchus, Tylodorus, Xiphinema, and Zygotylenchus nematode.
 26. Themethod of claim 23, said method further comprising providing in the dietof said pest a pesticidally effective amount of one or more other toxicagents selected from the group consisting of methylketone synthase,dsRNA, a Cry protein, a VIP protein, and a chemical nematicide.
 27. Themethod of claim 23, said method further comprising providing in a dietof said pest a pesticidally effective amount of one or more pesticidalpolypeptides, wherein said one or more pesticidal polypeptides compriseSEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ II) NO:18, SEQID NO:22, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:38, SEQ IDNO:46, SEQ ID NO:50, SEQ ID NO:54, SEQ ID NO:58, or SEQ ID NO:60, or apesticidal fragment thereof.
 28. The method of claim 27, wherein thepesticidally effective amount of said one or more pesticidalpolypeptides is provided by the plant, which is a recombinant plant, apart of the plant, or a product of the plant or the plant part.
 29. Themethod of claim 27, wherein the pesticidally effective amount of saidone or more pesticidal polypeptides is provided in one or moreformulations topically applied on the plant or a part of the plant, saidone or more formulations comprising bacterial cells, spores, orparasporal crystals that comprise said one or more pesticidalpolypeptides.